1. Technical Field
Apparatus and methods for high-throughput formation of an array of deposits on a substrate with improved resolution and pattern fidelity are disclosed. In particular, this disclosure relates to the manufacture and use of a removable stencil aligned with a high-throughput deposition printer to control the location and dimension of a single, multiplexed, or combinatorial array of deposits on the substrate.
2. Description of the Related Art
Methods and apparatus for depositing arrays of substances with smaller dimensions (i.e. micro- or even nano-scales) on a substrate have been the central focus of a number of technological fields including electronics, optics, chemistry and biochemistry. In particular, biomolecular arrays with nano-scale features are useful for applications such as tissue engineering, cell cultures, and studying subcellular receptor-ligand interactions and intracellular processes. For example, cell behavior such as differentiation, adhesion and proliferation, can be regulated by patterned arrays of extracellular matrix (ECM) proteins with different feature shapes, sizes and spacings. Reducing the feature size of patterned ECM proteins to sub-200 nm dimension can help to elucidate the role of ECM proteins in forming focal adhesions with single-molecule resolution.
Furthermore, there is a growing interest to integrate biological and chemical functionalities with miniaturized sensor devices whereby accurate spatial positioning and alignment are crucial, such as nano-wire sensors, chemical field-effect transistors, nano-electromechanical sensors, and diffraction based antibody gratings. The diversity of protein molecules and their combinations present in nature requires the highly multiplexed capability of arrays to study the plethora of possible antagonistic and synergistic interactions between receptors and ligands. Hence, it is typical for hundreds or thousands of different biomaterial samples and their replicates to be patterned on a large area array and afterwards, allow for biomolecular ligands or cells to interact with the patterned surface.
Recent advances in printing and lithography have enabled the generation of patterned arrays with smaller features, some even with nano-scale resolutions. Exemplary array patterning techniques may include micro-contact printing (μCP) and atomic-force microscopy (AFM) based methods such as dip-pen lithography (DPN). While DPN is able to achieve nano-scale resolution down to tens of nm, the method is not easily scalable to print thousands of different biomolecules in a high-throughput fashion. Moreover, the final shape and size of deposits printed using DPN are controlled by surface hydrophobicity and chemistry. DPN may require extended length of time required to pattern large areas >1 cm2 even if multiple pens are used. μCP, on the other hand, may generate replicate arrays more rapidly but the elastomeric polymer stamps can deform with pressure and swell in aqueous conditions, resulting in relatively non-uniform nano-scale features. Additionally, μCP may not be suitable for generating arrays of superimposed depositions of multiple different types of samples as repeated alignment of elastomeric stamps with nano-scale precision may be difficult to achieve. Finally, the dimension, resolution, shape and reproducibility of deposits printed using current conventional printing methods depend on many factors such as surface chemistry, hydrophobicity and printing buffer and thus there is a need for robust and efficient printing apparatus and methods with better resolution and pattern fidelity.
Use of a polymer stencil in the deposition of micro-scale arrays is also known in the art. Specifically, a pre-defined array of micro-scale openings may be created photolithographically in a photoresist mask, and may be transferred into the polymer stencil through an etching process, which in turn is placed on the substrate for deposition of biomolecular samples through the micro-scale openings. Removal of the polymer stencil leaves the array of samples with defined micro-scale features on the substrate. Exemplary polymeric materials for making the stencil includes parylene, which is biocompatible and has been used as a template for patterning biomolecular arrays with features >1 μm, such as creating large area arrays of single-cells, proteins, nucleic acids, and lipid bilayers.
However, existing polymer stencil technologies typically rely on bath application of one type of sample onto the whole surface. Moreover, existing polymer stencil technologies may also be limited by the dimensions of the openings created in the stencil. Finally, high-throughput deposition of multiplexed combinatorial samples using polymer stencil technologies has yet to be developed.